The assembly of ribonucleoprotein (RNP) complexes into microscopically visible granules occurs as part of the normal cellular regulation of RNA metabolism (processing bodies) and in response to stress (stress granules). Large RNP granules are also induced in the germline by stress or delayed fertilization. The long-term objectives of this application are to understand the function of RNPs in germ cells and resolve the question if they function as a cause or consequence of altered RNA metabolism. The overall approach is to investigate RNP function utilizing Caenorhabditis as an in vivo, multicellular model system, with the hypothesis that large RNP granules form in oocytes in order to maintain RNA stability, repress translation, and maintain overall oocyte viability/ fertility. The first specific aim of the proposal is to identify and characterize the genes required for assembly and dissociation of RNP granules. Microarray analyses will be performed to identify genes differentially expressed between worms with and without RNP granules present in oocytes. These genes and others previously identified as "germline intrinsic" will be targeted in a functional RNAi screen to identify genes required for the MEX-3 RNA-binding protein to assemble into large granules when ovulation is arrested. Characterization of the positive hits by immunofluorescence experiments will determine whether each gene regulates MEX-3 localization to granules specifically or controls more global RNP granule assembly. The effect of microtubule disruption, the dependence on eIF2a, and the role of polysome stability in RNP granule assembly will be determined using a combination of inhibitor microinjections and targeted RNAi experiments. The second specific aim is to directly test the hypothesis for RNP granule function. Fertility will be assessed after mating into worms with disrupted RNP granule formation in oocytes. RNA stability and translational repression will similarly be determined using qRT-PCR and immunofluorescence experiments, respectively. The third specific aim includes an ultrastructural characterization of germline RNPs to determine if nuclear pores, mitochondria, and ribosomes are differentially localized in germlines when ovulation is arrested or stress is present. Taken together, these studies will provide significant insight into the regulation and function of RNPs in germ cells. The results may have relevance to understanding the basis for infertility that occurs as women age and the mechanisms and general strategies cells employ when stresses occur. PUBLIC HEALTH RELEVANCE: The broad goal of this project is to gain insight into the mechanisms used by cells to regulate protein translation during stress. Large complexes of RNA and RNA-binding proteins (RNPs) accumulate into stress granules to regulate translation in response to stress, and similar RNP granules assemble in eggs (oocytes) when they must wait a prolonged period to be fertilized or undergo an environmental stress. This research tests the hypothesis that large RNP granules play a protective role in maintaining oocyte viability and fertility and may be relevant to understanding the basis for infertility that occurs as women age.